In this era of increasing resistance to current antibiotics, novel, innovative approaches to antimicrobial development are needed. New paradigms for antibiotic therapy will be contingent upon a better understanding of the basic mechanisms of in vivo pathogenesis and the complex relationship between the host and pathogen. The goal of this project is to merge the powerful fields of chemical biology and genomics and apply them to the study of Vibrio cholerae virulence regulation. We propose to identify and investigate new regulatory pathways that control virulence expression in Vibrio cholerae, the Gram-negative bacterium that causes the human diarrheal disease called cholera. As a model organism, it is a genetically tractable system for understanding bacterial pathogenesis, as evidenced by the previous successful identification of some of its virulence factors and requirements for colonization and infection. Yet, much remains unknown with regards to the organism's mechanisms to sense and respond to virulence activating stimuli within the host microenvironment. Thus, it also represents a model system for studying bacterial pathogenesis and uncovering underlying principles in pathogen response to the host microenvironment. We propose to use novel small molecules that we have identified in a high-throughput chemical screen for inhibitors of virulence gene expression in V. cholerae to probe and study in vivo regulatory mechanisms. Preliminary studies of these compounds suggest that they define novel regulatory steps that have not been previously described, including steps that involve sensing of low oxygen tension and that coregulate virulence expression and biofilm formation. We will complement these chemical biological studies with genomic approaches, including the systematic identification of genes involved in virulence regulation pathways, using comprehensive libraries that either knock-out every gene (except in vitro essential genes) or overexpress every gene. PUBLIC HEALTH RELEVANCE: The goal of this project is to elucidate novel pathways of virulence regulation in the pathogen Vibrio cholerae and to understand how its interaction with the host triggers a bacterial program that ultimately results in disease and host damage. This project not only serves as a model to advance our understanding of host-pathogen interactions and the infectious disease process in general, but also provides new paradigms for therapeutic intervention based on anti-virulence approaches.